The protocol helps the students identify individual infections clearly and perform the assay easily, making it more efficient to quantify viral titer. This protocol incorporates a simple fixation and staining procedure that produces reliable and reproducible results. This method can provide insight into the accuracy and effectiveness of antiviral treatments against diseases.
The more difficult hurdles of this plaquing protocol are the cell counts for seeding plates, making sure that the cells do not overcrowd, dry out, or get scratched throughout the process. One may also find difficulty in the overall meticulous nature of this protocol. However, practice is a major contributor of the success of this experiment.
To begin, complete the sample dilution in the addition of virus to cells in one lab session. Serially dilute each virus sample being plaqued in PBS. Typically use 10-fold dilutions for most precise tracking.
Keep all the viral dilutions on ice until ready to add these diluted virus samples to the cells. Remove the cell culture medium from one or two wells using a pipette. Carefully add the diluted virus sample dropwise to each monolayer of cells dropwise through the side of each well.
Repeat the process for every one or two wells until the entire plate is filled with the virus samples being plaqued. Gently rock the entire plate by hand. Then incubate the plate at 37 degree Celsius for one hour to allow the virus adsorption.
After every 10 minutes, remove the plate from the incubator, gently rock it again to spread the virus more evenly across each well, and then place it back in the incubator. To diminish the possibility of inadvertently counting unabsorbed inoculum, remove the virus sample from the wells using a 1000-microliter pipette tip and discard the sample in a waste beaker. Place 2.5 milliliters of a methylcellulose overlay on the plate, and place it back in the incubator.
Disinfect the waste beaker, typically with the addition of bleach, an iodophor, or a similar virucide, before removing it from the biosafety cabinet. After the two-day incubation, remove the methylcellulose overlay from one or two wells at a time using a pipette, and place it in a waste beaker. Add approximately two milliliters of 1%crystal violet in 50%ethanol to each well.
Incubate the plate with all wells filled with the stain for 30 minutes at 37 degrees Celsius. At this point, disinfect the waste beaker as demonstrated previously. Wash the plates vigorously with tap water until the runoff is clear.
Ensure that there are approximately 10-fold differences in the number of plaques across each dilution series. The figure depicts a 10-fold decrease in plaque counts where the countable number of plaques fell in the 10 to the minus four range for all three replicates. The first three images in the figure depict plaques in the 10 to the minus three dilution.
However, the bottom row shows results when a plaque assay is not conducted well. There are very few plaques visible from any dilution in the visible areas around the top where the Vero cells have entirely lifted from the substrate. Similarly, this figure shows problems with cells drying out or mishandling during the plaque assay.
However, this figure critically shows poor Vero cell seeding. Every well shows darker purple spots, indicative of cells not spread well across the well and piled on top of one another in clusters. For best results, make sure the virus is spread uniformly across the plate to avoid clumped up plaques Plaque assays are more effective than other quantitative methods because it only counts live virus.
That way, true antiviral efficacy can be established. Additionally, this technique has allowed us to quantify viral presence on fomites and explore the effectiveness of drug delivery from novel medical devices within our sector of herpes virus research.
